RE: Sorting big cells on FACSAria!

One of the PI's at my old lab had the great idea of nuking the AccuDrop
beads in a cobalt irradiation source to sterilise them so that we could slip
them in at any point in the sort without adding (viable) bugs - it worked
very well.
I'd imagine that any x-ray/gamma irradiator could be used to clean up beads
of any kind if you're lucky enough to have access to one (the irradiator we
had was mainly used for preparing feeder cells for long term culture, but
wasn't too different to those used for sterilizing filters and other labware
commercially).

Cheers

Ray


-----Original Message-----
From: vhodara@sfbr.org [mailto:vhodara@sfbr.org] 
Sent: Wednesday, May 07, 2008 12:36 AM
To: cyto-inbox
Subject: Sorting big cells on FACSAria!

Dear experts in flow sorting:

I am trying to sort HUH7 cells GFP (brigth and not so bright populations)
Just
two gates in a single color FL1 vs SSC (very simple sorting should be).
Those
are big cells so I tried with the 70um nozzle but with medium pressure. I
haven't had problems with settings, or with accudrop. However, I couldn't
have
pure populations. When setting the streams I can see the two perfect dots on
the camera on the screen and accudrop run perfectly, but when I am sorting I
don't see but a smear of white dots to the right and to the left of the main
stream (is that because of the big cells?). I am planning to try the 100um
nozzle, but can I run the accudrop with this nozzle eventhough they are very
small? If somebody can give me some tips, I will be very grateful.
And another question, if I prepare the machine for asceptic sorting, can I
run
the accudrop before or I need to prepare a sterile solution of these beads
or
run them after asceptic cleaning and then run again bleach and water for a
few
minutes to keep the "sterility". (We do not have the FACSAria in a BSL-3 lab
yet)
Thanks a Lot
Vida